Heterozygosity and Chain Multivalents during Meiosis Illustrate Ongoing Evolution as a Result of Multiple Holokinetic Chromosome Fusions in the Genus Melinaea (Lepidoptera, Nymphalidae).

Mitotic and meiotic chromosomes from 2 taxa of the genus Melinaea, M. satevis cydon and M. "satevis" tarapotensis (Lepidoptera: Nymphalidae), and from hybrids produced in captivity were obtained using an improved spreading technique and were subsequently analyzed. In one of the taxa, the presence of trivalents and tetravalents at diakinesis/metaphase I is indicative of heterozygosity for multiple chromosome fusions or fissions, which might explain the highly variable number of chromosomes previously reported in this genus. Two large and complex multivalents were observed in the meiotic cells of the hybrid males (32 chromosomes) obtained from a cross between M. "s." tarapotensis (28 chromosomes) and M. s. cydon (40-43 chromosomes). The contribution of the 2 different haploid karyotypes to these complex figures during meiosis is discussed, and a taxonomic revision is proposed. We conclude that chromosome evolution is active and ongoing, that the karyotype of the common ancestor consisted of at least 48 chromosomes, and that evolution by chromosome fusion rather than fission is responsible for this pattern. Complex chromosome evolution in this genus may drive reproductive isolation and speciation, and highlights the difficulties inherent to the systematics of this group. We also show that Melinaea chromosomes, classically considered as holocentric, are attached to unique, rather than multiple, spindle fibers.

Molecular properties of the N-terminal extension of the fission yeast kinesin-5, Cut7.

Kinesin-5 plays an essential role in spindle formation and function, and serves as a potential target for anti-cancer drugs. The aim of this study was to elucidate the molecular properties of the N-terminal extension of the Schizosaccharomyces pombe kinesin-5, Cut7. This extension is rich in charged amino acids and predicted to be intrinsically disordered. In S. pombe cells, a Cut7 construct lacking half the N-terminal extension failed to localize along the spindle microtubules and formed a monopolar spindle. However, a construct lacking the entire N-terminal extension exhibited normal localization and formed a typical bipolar spindle. In addition, in vitro analyses revealed that the truncated Cut7 constructs demonstrated similar motile velocities and directionalities as the wild-type motor protein, but the microtubule landing rates were significantly reduced. These findings suggest that the N-terminal extension is not required for normal Cut7 intracellular localization or function, but alters the microtubule-binding properties of this protein in vitro.

The nuclear mitotic apparatus (NuMA) protein: localization and dynamics in human oocytes, fertilization and early embryos.

The oocyte's meiotic spindle is a dynamic structure that relies on microtubule organization and regulation by centrosomes. Disorganization of centrosomal proteins, including the nuclear mitotic apparatus (NuMA) protein and the molecular motor complex dynein/dynactin, can lead to chromosomal instability and developmental abnormalities. The present study reports the distribution and function of these proteins in human oocytes, zygotes and early embryos. A total of 239 oocytes, 90 zygotes and discarded embryos were fixed and analyzed with confocal microscopy for NuMA and dynactin distribution together with microtubules and chromatin. Microtubule-associated dynein-dependent transport functions were explored by inhibiting phosphatase and ATPase activity with sodium-orthovanadate (SOV). At germinal vesicle (GV) stages, NuMA was dispersed across the nucleoplasm. After GV breaks down, NuMA became cytoplasmic before localizing at the spindle poles in metaphase I and II oocytes. Aberrant NuMA localization patterns were found during oocyte in vitro maturation. After fertilization, normal and abnormal pronuclear stage zygotes and embryos displayed translocation of NuMA to interphase nuclei. SOV treatment for up to 2 h induced lower maturation rates with chromosomal scattering and ectopic localization of NuMA. Accurate distribution of NuMA is important for oocyte maturation, zygote and embryo development in humans. Proper assembly of NuMA is likely necessary for bipolar spindle organization and human oocyte developmental competence.

Spindle and chromosome configurations of in vitro-matured oocytes from polycystic ovary syndrome and ovulatory infertile women: a pilot study.

PURPOSE: To evaluate the meiotic spindle and chromosomal distribution of in vitro-matured oocytes from infertile nonobese women with PCOS and male or tubal causes of infertility (controls), and to compare in vitro maturation (IVM) rates between groups. METHODS: Seventy four patients (26 with PCOS and 48 controls) undergoing stimulated cycles of oocyte retrieval for ICSI were selected prospectively. Thirteen PCOS patients and 27 controls had immature oocytes retrieved submitted to IVM. After IVM, oocytes showing extrusion of the first polar body were fixed and processed for evaluation of the meiotic spindle and chromosome distribution by immunofluorescence microscopy. RESULTS: There were no differences between PCOS and control groups with respect to IVM rates (50.0% and 42.9%, respectively) nor the percentage of meiotic abnormalities in metaphase II oocytes (35.3% and 25%, respectively). CONCLUSIONS: In vitro-matured oocytes obtained from stimulated cycles of nonobese PCOS did not have an increased ratio of meiotic abnormalities.

Effects of in vitro maturation and age on oocyte quality in the rhesus macaque Macaca mulatta.

OBJECTIVE: To evaluate oocyte quality in a primate model. DESIGN: Analysis of oocyte karyotype by chromosome spreading and oocyte spindles by confocal microscopy. SETTING: Research laboratory, Caribbean Primate Research Center. ANIMAL(S): Rhesus macaques aged 6-22 years. INTERVENTION(S): Fourteen females underwent both Regimen A (FSH + hCG) and Regimen B (FSH only) stimulation cycles to facilitate collection of mature and immature oocytes. Immature oocytes from Regimens A and B underwent in vitro maturation (IVM) to produce metaphase II oocytes. All metaphase II oocytes underwent gradual fixation to spread chromosomes or were fixed and stained with probes specific to alpha-tubulin, actin, and DNA for visualization of the meiotic spindle using confocal microscopy. MAIN OUTCOME MEASURE(S): Karyotype and meiotic spindle architecture differences among in vivo matured (IVO) and IVM oocytes from young and old rhesus macaques. RESULT(S): In all, 4.7% of IVO oocytes (Regimen A) from young females were hyperhaploid versus 25.0% of IVM oocytes (Regimen B) from old females; 4.5% of IVO oocytes (Regimen A) from young females versus 51.5% of IVM oocytes (Regimen B) from old females displayed abnormal chromosome alignment on the metaphase spindle. CONCLUSION(S): IVM can induce meiotic anomalies in macaque oocytes, especially those obtained from older females. Results from this study provide possible explanations for the reported reduction in developmental competence of IVM primate oocytes.

Zona pellucida birefringence score and meiotic spindle visualization in relation to embryo development and ICSI outcomes.

The meiotic spindle and the zona pellucida exhibit molecular order when imaged with polarized optics. This study aimed to investigate possible factors contributing to the zona pellucida birefringence score and meiotic spindle visualization, and to evaluate whether these structures may predict intracytoplasmic sperm injection outcomes. Oocytes were divided into groups according to zona pellucida birefringence and meiotic spindle visualization. In addition, the cycles were split into three groups based on the zona birefringence of transferred embryos. A positive correlation was observed between zona birefringence and meiotic spindle visualization. In addition, when the meiotic spindle was observed, the fertilization rate among oocytes with high or low zona pellucida birefringence was similar. Implantation and pregnancy rates were significantly higher when embryos derived from high birefringence oocytes were exclusively transferred (P = 0.041 and P = 0.004 respectively). Furthermore, the miscarriage rate was higher when embryos derived from low birefringence oocytes were exclusively transferred. On the other hand, the total dose of FSH negatively affected meiotic spindle visualization. Results show that selection of embryos based on zona pellucida and meiotic spindle imaging can significantly improve implantation and pregnancy rates. Moreover, the dose of FSH used for ovarian stimulation may affect the organization of the oocyte meiotic spindle.

Relationship between visualization of meiotic spindle in human oocytes and ICSI outcomes: a meta-analysis.

The objective of this meta-analysis was to investigate the influence of meiotic spindle visualization in human oocytes on intracytoplasmic sperm injection (ICSI) outcomes. Search strategies included on-line surveys of databases (MEDLINE, EMBASE, Science Citation Index, Cochrane Controlled Trials Register and Ovid). The fixed effect was used for odds ratio. Ten trials fulfilled the inclusion criteria comparing in-vitro and clinical ICSI outcomes with or without visualization of meiotic spindle in fresh and in-vivo matured oocytes. According to the meta-analysis, the results showed statistically significant higher fertilization rate (P < 0.0001) when the meiotic spindle was viewed than when it was not. Moreover, the percentage of pro-nuclear-stage embryos with good morphology (P = 0.003), cleavage rate (P < 0.0001), percentage of day-3 top-quality embryos (P = 0.003) and percentage of embryos that reached the blastocyst stage (P < 0.0001) were statistically significantly better among embryos derived from oocytes in which meiotic spindle was viewed compared with those in which meiotic spindle was not observed. However, these differences were not observed in the clinical pregnancy or implantation rates. This observation has clinical relevance mainly in countries where there is a legal limit on the number of oocytes to be fertilized. However, additional controlled trials are needed to further confirm these results.

Mitotic, but not meiotic, oriented cell divisions in rat spermatogenesis.

The process of mammalian spermatogenesis involves both mitosis and meiosis at the same developmental age. Most previous studies have focused on mitotic spindle orientation during development, but not during meiotic division. Therefore, we asked whether there is a difference between mitotic and meiotic germ cell spindle orientation during rat spermatogenesis. Our results showed that mitotic spindles of spermatogonia were mainly oriented with angles ranging from 60 to 90 degrees, perpendicular in relation to the basement membrane of the seminiferous tubules. On the other hand, meiotic spindles showed a random orientation. Nocodazole treatment (at a concentration that depolymerizes only astral microtubules) induced a significant increase in cells with an angle between 0 and 30 degrees (parallel) in relation to the basement membrane. Meiotic spindles did not show a significant change in their orientation after the Nocodazole treatment. Therefore, our results suggest differences between the mechanisms controlling positioning and orientation of mitotic and meiotic spindles during rat spermatogenesis. It seems that a phylogenetically conserved programme controls the mitotic spindle orientation in organisms ranging from worms to mammals.

Spindle imaging: a marker for embryo development and implantation.

OBJECTIVE: To examine whether the presence of birefringent spindles in living human oocytes can be used as a predictive factor associated with embryo morphology to allow embryo selection before transfer and its association with IVF outcomes. DESIGN: Prospective study. SETTING: Assisted reproduction center in Brazil. PATIENT(S): One hundred fifty-seven patients undergoing intracytoplasmic sperm injection cycles, resulting in 1,097 metaphase II oocytes. INTERVENTION(S): Meiotic spindles were evaluated before intracytoplasmic sperm injection in all metaphase II oocytes. MAIN OUTCOME MEASURE(S): Meiotic spindles' imaging and fertilization rate, embryo development, and implantation rate. RESULT(S): Birefringent spindles were detected in 65.9% (SD group). The normal fertilization rate and rate of early-cleavaged embryos were higher in the SD group compared with in the spindle-non-detected (SND) group. When only embryos from the SD group were selected for transfer, the pregnancy and implantation rates were 44.4% and 23.0%, and when only embryos from the SND group were transferred, those rates were 18.2% and 8.7%, respectively (statistically significant differences). CONCLUSION(S): Spindle visualization can be an important tool for predicting better fertilization potential, embryo development, and clinical outcomes, suggesting that embryo selection for transfer may be based not only on embryo morphology but also on oocyte nuclear maturity.

Prognostic value of meiotic spindle imaging on fertilization rate and embryo development in in vitro-matured human oocytes.

OBJECTIVE: To evaluate the relationship between spindle visualization and intracytoplasmic sperm injection (ICSI) outcomes in controlled ovarian stimulation (COS) cycles. DESIGN: Prospective study. SETTING: Assisted reproduction center. PATIENT(S): Thirty patients undergoing ICSI cycles. INTERVENTION(S): Meiotic spindle was evaluated before ICSI in 234 in vivo- and 101 in vitro-matured oocytes MAIN OUTCOME MEASURE(S): Meiotic spindle imaging, fertilization rate, and embryo development. RESULT(S): Spindle was present in 74.3% and 73.8% of the in vivo- and in vitro-matured oocytes, respectively. Spindle detection rate in oocytes derived from germinal vesicle and metaphase-I stage was, respectively, 50% and 86%. The fertilization rate achieved by the in vivo-matured oocytes was 71.8%, and spindle was detected in 75.6% of the fertilized oocytes and only 34.8.% of the nonfertilized oocytes. In the in vitro-matured oocytes, the fertilization rate was 66.1%, and spindle was detected in 81.4% of the fertilized oocytes and in 59.1% of the nonfertilized oocytes. Ten out of 43 (23.2%) in vitro-matured derived embryos were considered to be high quality, all derived from spindle-detected oocytes, which represents an increase of 13.0% on the overall number of high-quality embryos. CONCLUSION(S): Meiotic spindle imaging may be useful to predict in vitro-matured oocyte development. However, other factors may contribute to the decreased developmental competence of in vitro-matured oocytes.

The mitotic spindle and associated membranes in the closed mitosis of trichomonads.

In the present work, we followed the several phases of Tritrichomonas foetus and Trichomonas vaginalis cell cycles using immunofluorescence, serial thin sections, three-dimensional (3D) reconstruction, and transmission electron microscopy. In motile trichomonad cells or in pseudocyst forms, the nuclear envelope persists throughout mitosis, and the spindle is extranuclear. We found three types of spindle microtubules: pole-to-nucleus microtubules which are attached to the nuclear envelope, pole-to-pole microtubules forming a cylindrical, cytoplasmic groove on the nuclear compartment in pseudocysts of T. foetus cells, and pole-to-cytosol microtubules which extend freely into the cytoplasm. We demonstrated that: (1) in T. foetus, the spindle is assembled from an MTOC located at the base of the costa, underneath one of the basal bodies; (2) the spindle presents an unusual arc shape during the initial phases of mitosis in motile trophozoites; (3) the spindle microtubules are glutamylated, but not acetylated; (4) several membranes similar to those of the endoplasmic reticulum follow the spindle microtubules; (5) finger-like projections extend from the nucleus towards the cell poles in pseudocysts and multinucleated cells; and (6) vesicles formed in between the two nuclear membranes are seen in the course of mitosis in both trophozoite and pseudocyst forms.

Contributions of the axostyle and flagella to closed mitosis in the protists Tritrichomonas foetus and Trichomonas vaginalis.

Tritrichomonas foetus and Trichomonas vaginalis are protists that undergo closed mitosis: the nuclear envelope remains intact and the spindle remains extranuclear. Here we show, in disagreement with previous studies, that the axostyle does not disappear during mitosis but rather actively participates in it. We document the main structural modifications of the cell during its cell cycle using video enhanced microscopy and computer animation, bright field light microscopy, confocal laser scanning microscopy, and scanning and transmission electron microscopy. We propose six phases in the trichomonad's cell cycle: an orthodox interphase, a pre-mitotic phase, and four stages during the cell division process. We report that in T. foetus and T. vaginalis: a) all skeletal structures such as the costa, pelta-axostyle system, basal bodies, flagella, and associated filaments of the mastigont system are duplicated in a pre-mitotic phase; b) the axostyle does not disappear during mitosis, otherwise playing a fundamental role in this process; c) axostyles participate in the changes in the cell shape, contortion of the anterior region of the cell, and karyokinesis; d) flagella are not under assembly during mitosis, as previously stated by others, but completely formed before it; and e) cytokinesis is powered in part by cell locomotion.

Cytoskeletal organization defects and abortive activation in human oocytes after IVF and ICSI failure.

In this study, we analysed the distribution of beta tubulins to detect spindle and cytoplasmic microtubules, alpha acetylated tubulins for sperm microtubules and chromatin configuration in oocytes showing fertilization failure after conventional IVF or intracytoplasmic sperm injection (ICSI). A total of 450 human oocytes that failed to fertilize were studied 20-40 h after IVF or ICSI. In all, 287 oocytes were stained for immunofluorescence and chromosomal spreads were performed by Tarkowski's air-drying method in 163 IVF or ICSI oocytes that did not develop pronuclei after the extrusion of a second polar body. Immunofluorescence analysis showed that the main reason of fertilization failure after IVF was no sperm penetration (55.5%). The remaining oocytes showed different abnormal patterns, e.g. oocyte activation failure (15.1%) and defects in pronuclei apposition (19.2%). On the other hand, fertilization failure after ICSI was mainly associated to incomplete oocyte activation (39.9%), and to a lesser extent with defects in pronuclei apposition (22.6%) and failure of sperm penetration (13.3%). A further 13.3% of the ICSI oocytes arrested their development at the metaphase of the first mitotic division. The chromosomal spreads allowed the analysis of abortive activations, in which no pronuclei formed but a second polar body was extruded. Immunofluorescence and cytogenetic analysis provided a useful tool to improve infertility diagnosis and prognosis in each particular case.

Entamoeba histolytica: microtubule movement during mitosis.

The movement of microtubules (MTs) during nuclear division of Entamoeba histolytica was ultrastructurally studied. Regarding this MT movement, five stages of mitosis could be defined: prophase, metaphase, anaphase A, anaphase B, and telophase. In early stages of mitosis, chromatinic material appeared condensed, and MTs were detected in the center of the nucleus. Later, MTs seemed to grow from an electron-dense body located in the center of the nucleus. This body might be the microtubule organizing center, which organized the MTs, first in a lateral way, and later to form the mitotic spindle, which was made of a bundle of MTs joined by their ends. This junction of MTs to themselves could also be observed in cross-sections. The last stage of mitosis was the nuclear separation. Two different morphological types of intranuclear vesicles were also observed, which seemed to have different types of membrane. Both intranuclear vesicles were present during nuclear division, generally in clusters, and located close to the nuclear periphery.

[Ultrastructure of the mitotic nuclei in Leishmania mexicana ssp. Tridimensional reconstruction of the mitotic spindle and dense plaques]. / Ultraestructura del núcleo mitótico en Leishmania mexicana ssp. Reconstrucción tridimensional del huso mitótico y placas densas.

The ultrastructure of mitotic nuclei of the promastigote Leishmania mexicana ssp. was studied by serial thin sections and three-dimensional reconstructions of each divisional stage. At the beginning of nuclear division (equatorial stage), a set of six dense plaques located about the equatorial region of the nucleus and a microtubular spindle develops in the two opposing poles of the nucleus (two sets of polar microtubules). The microtubular mitotic spindle is entirely intranuclear with the nuclear membrane persisting through mitosis. The polar spindle consists of a discrete bundle of about 50 microtubules and the equatorial spindle is formed by about 100 microtubules. The spindle may contain several continuous microtubules, but no microtubular organizing centres were observed in association with the spindle. The plaques and hemiplaques are associated with microtubular bundles; some of the spindle microtubules converge on kinetochore-like plaques. It is suggested that the spindle has a special significance in the physiology of mitosis. The two sets of hemiplaques may guide the separation of the daughter genomes. At the beginning of the elongational stage the mitotic plaques split into halves and each set of half-plaques migrates to one pole. It is concluded that the dense plaques play a kinetochore-like role and thus Leishmania mexicana ssp. may have six chromosomal units. Mitotic events of this species are essentially similar to those of Trypanosoma cruzi.

Ultraestructura del núcleo mitótico en Leishmania mexicana ssp.: reconstrucción tridimensional del huso mitótico y placas densas / Ultrastructure of the mitotic nuclei in Leishmania mexicana ssp.: tridimensional reconstruction of the mitotic spindle and dense plaques

Se estudió mediante cortes ultrafinos seriados, la ultraestructura del núcleo mitótico en una especie del complejo Leishmania mexicana. Al inicio de la división nuclear, un grupo de seis placas densa se localiza en la región ecuatorial del núcleo y un huso microtubular se forma entre dos polos opuestos. El huso mitótico es completamente intranuclear, con la membrana nuclear presente en todo el proceso de la división. Los huso polares están formados por aproximadamente (zona de superposición) por aproximadamente 100 microtúbulos. No se observó centros organizadores de microtúbulos en relación con el huso. Las placas y hemiplacas apareciaron en asociación con grupos de microtúbulos, que finalizan en ellas o pasan tangencialmente. Esto sugiere que el huso tiene un especial significado en la ffisiologia del desplazamiento de las hemiplacas durante la separación de los genomios. Al inicio del estado de elongación, las placas se dividen en mitades y cada grupo emigra a un polo opuesto. Se concluye que las placas juegan un papel similar a de los cinetocoros y así Leishmania mexicana tendría seis unidades cromosomales. Los eventos mitóticos en esta especie son esencialmente similares a los observados en Trypanosoma cruzi

Ultrastructure of the mitotic nucleus in Leishmania mexicana sp. tridimensional reconstruction of the mitotic sindle and dense plaques

Se ha estudiado mediante cortes ultrafinos seriados la ultraestructura del núcleo mitótico en una especie del complejo Leishmania mexicana. Cambios en el núcleo interfásico y en los cuatro estados de la división son descritos. Al inicio de la división nuclear, un grupo de seis placas densas se localizan en la región ecuatorial del núcleo y un huso microtubular se forma entre dos polos opuestos. El huso mitótico es completamente intranuclear, con las membrana nuclear presente en todo el proceso de la división. Los husos polares están formados por aproximadamente 50 microtúbulos, y el ecuatoial (zona de superposición) por aproximadamente 100 mucrotúbulos. No se observaron centros organizadores de microtúbulos en relación con el huso. Las placas y hemiplacas fueron obaservadas en asociación con grupos de microtúbulos, que finalizan en ellas o pasan tangencialmente. Esto sugiere que el Huso tiene un especial significado en la fisiología del desplazamiento de las hemiplacas durante la separación de los genomios. Al inicio del estado elongacional, las placas se dividen en mitades y cada grupo emigra a un polo opuesto. Se concluye que las placas juegan un papel similar al de los cinetocoros y así Leishmania mexicana tendría seis unidades cormosomales. Los eventos mitóticos en esta especie son esencialmente similares a los observados en Trypanosoma cruzi

Ultrastructure of the mitotic nucleus in Leishmania mexicana sp. tridimensional reconstruction of the mitotic sindle and dense plaques

Se ha estudiado mediante cortes ultrafinos seriados la ultraestructura del núcleo mitótico en una especie del complejo Leishmania mexicana. Cambios en el núcleo interfásico y en los cuatro estados de la división son descritos. Al inicio de la división nuclear, un grupo de seis placas densas se localizan en la región ecuatorial del núcleo y un huso microtubular se forma entre dos polos opuestos. El huso mitótico es completamente intranuclear, con las membrana nuclear presente en todo el proceso de la división. Los husos polares están formados por aproximadamente 50 microtúbulos, y el ecuatoial (zona de superposición) por aproximadamente 100 mucrotúbulos. No se observaron centros organizadores de microtúbulos en relación con el huso. Las placas y hemiplacas fueron obaservadas en asociación con grupos de microtúbulos, que finalizan en ellas o pasan tangencialmente. Esto sugiere que el Huso tiene un especial significado en la fisiología del desplazamiento de las hemiplacas durante la separación de los genomios. Al inicio del estado elongacional, las placas se dividen en mitades y cada grupo emigra a un polo opuesto. Se concluye que las placas juegan un papel similar al de los cinetocoros y así Leishmania mexicana tendría seis unidades cormosomales. Los eventos mitóticos en esta especie son esencialmente similares a los observados en Trypanosoma cruzi (AU)